0009CCB

Current Chemical Biology, 2009, 3, 203-230 203
1872-3136/09 $55.00+.00 2009 Bentham Science Publishers Ltd.

Functional Characterization of Chitin and Chitosan
Inmaculada Aranaz, Marian Mengbar, Ruth Harris, Ins Paos, Beatriz Miralles, Niuris Acosta,
Gemma Galed and ngeles Heras*
Department of Physical Chemistry II, Faculty of Pharmacy, Institute of Biofunctional Studies, Complutense University,
Paseo Juan XXIII, n 1. Madrid 28040, Spain
Abstract: Chitin and its deacetylated derivative chitosan are natural polymers composed of randomly distributed -(1-4)-
linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). Chitin is insoluble in aqueous
media while chitosan is soluble in acidic conditions due to the free protonable amino groups present in the D-glucosamine
units. Due to their natural origin, both chitin and chitosan can not be defined as a unique chemical structure but as a fam-
ily of polymers which present a high variability in their chemical and physical properties. This variability is related not
only to the origin of the samples but also to their method of preparation. Chitin and chitosan are used in fields as different
as food, biomedicine and agriculture, among others. The success of chitin and chitosan in each of these specific applica-
tions is directly related to deep research into their physicochemical properties. In recent years, several reviews covering
different aspects of the applications of chitin and chitosan have been published. However, these reviews have not taken
into account the key role of the physicochemical properties of chitin and chitosan in their possible applications. The aim
of this review is to highlight the relationship between the physicochemical properties of the polymers and their behaviour.
A functional characterization of chitin and chitosan regarding some biological properties and some specific applications
(drug delivery, tissue engineering, functional food, food preservative, biocatalyst immobilization, wastewater treatment,
molecular imprinting and metal nanocomposites) is presented. The molecular mechanism of the biological properties such
as biocompatibility, mucoadhesion, permeation enhancing effect, anticholesterolemic, and antimicrobial has been up-
dated.
Keywords: Chitin, chitosan, molecular weight, deacetylation degree, crystallinity, functional characterization.
1. INTRODUCTION
Among the novel families of biological macromolecules,
whose relevance is becoming increasingly evident, are chitin
and its main derivative, chitosan. Potential and usual applica-
tions of chitin, chitosan and their derivatives are estimated to
be more than 200 [1]. This wide range of applications in-
cludes biomedicine, food, biotechnology, agriculture and
cosmetics, among others. The importance of chitin and chi-
tosan in the last years is evident in Table 1.
Chitin and chitosan are described as a family of linear
polysaccharides consisting of varying amounts of (14)
linked residues of N-acetyl-2 amino-2-deoxy-D-glucose (de-
noted in this review as A residues) and 2-amino-2-deoxy-D-
glucose residues (denoted in this review as D residues). Chi-
tin samples have a low amount of D units and hence the
polymer is insoluble in acidic aqueous media (Fig. 1a). On
the other hand, the amount of D units in chitosan samples is
high enough to allow the polymer to dissolve in acidic aque-
ous media. Some authors consider that chitosan is the poly-
mer with at least 60% of D residues [2]. Chitin is the second
most abundant natural polymer in nature after cellulose and
it is found in the structure of a wide number of invertebrates
(crustaceans exoskeleton, insects cuticles) and the cell
walls of fungi, among others. On the other hand, chitosan
only occurs naturally in some fungi (Mucoraceae) [3].

*Address correspondence to this author at the Institute of Biofunctional
Studies, Complutense University, Paseo J uan XXIII, n 1, Madrid 28040,
Spain; Tel/Fax: +34-913943284;
E-mail: aheras@farm.ucm.es; inma@ieb.ucm.es
Chitosan can also be prepared by cleavage of N-acetyl
groups of the chitin A residues. This reaction is rarely con-
ducted to full completion; hence chitosan polymeric chain is
generally described as a copolymeric structure comprised of
D-glucosamine (D residues) along with N-acetyl residues
(Fig. 1b).
The fine structure of chitosan is defined by the overall or
bulk content of D-hexosamine residues as well as their dis-
tribution along the polymeric chain. The molar fraction of
residual A groups in chitosan is expressed as a degree of N-
acetylation (DA) or fraction of acetylation (F
a
). The molar
fraction of D residues, deacetylation degree (DD), is also
very frequently used.
In contrast to chitin, the presence of free amine groups
along the chitosan chain allows this macromolecule to dis-
solve in diluted aqueous acidic solvents due to the protona-
tion of these groups, rendering the corresponding chitosan
salt in solution. So, there are important experimental vari-
ables that should be taken into account when working with
chitosan solutions such as the nature of the salt counterion,
degree of acetylation, Mw, pH, ionic strength and the addi-
tion of a non-aqueous solvent.
The aim of the present review is to present a state-of-the-
art study of the relationship between the physico-chemical
properties of these two polymers and their biological activi-
ties, as well as their applications. Since this aim is very am-
bitious, due to the extension of the topic, chitin and chitosan
derivatives are not considered.
The review has been divided into the following sections:
the first part is devoted to the preparation, characterization,
204 Current Chemical Biology, 2009, Vol. 3, No. 2 Aranaz et al.
effects of the preparation process on the properties of chitin
and chitosan and regulatory aspects. The second part covers
the main biological properties of the polymers and relates
these properties to the physicochemical characteristics. Fi-
nally, several applications of both polymers are reviewed
emphasizing the effect of the polymers characteristics on
these applications.

Fig. (1). Chemical structure of 100% acetylated chitin (A) and chi-
tosan (B).
2. METHODS OF PREPARATION
A schematic representation of the processes to prepare
chitin and chitosan from raw material is shown in Scheme 1.
2.1. Chitin Extraction
As mentioned above, chitin is present within numerous
taxonomic groups. However, commercial chitins are usually
isolated from marine crustaceans, mainly because a large
amount of waste is available as a by-product of food process-
ing. In this case, -chitin is produced while squid pens are
used to produce -chitin.
The structure of -chitin has been investigated more ex-
tensively than that of either the - or - form, because it is
the most common polymorphic form. Very few studies have
been carried out on - chitin. It has been suggested that -
chitin may be a distorted version of either - or -chitin
rather than a true third polymorphic form [3].
In chitin, the chains are arranged in sheets or stacks,
the chains in any one sheet having the same direction or
sense. In -chitin, adjacent sheets along the c axis have the
same direction; the sheets are parallel, while in -chitin adja-
cent sheets along the c axis have the opposite direction, they
are antiparallel .In - chitin, every third sheet has the oppo-
site direction to the two preceding sheets [3]. A schematic
representation of the three structures is shown in Fig. (2).

Scheme 1. Preparation of chitin and chitosan from raw material.
Crustacean shells consist of 30-40% proteins, 30-50%
calcium carbonate, and 20-30% chitin and also contain pig-
ments of a lipidic nature such as carotenoids (astaxanthin,
astathin, canthaxanthin, lutein and -carotene). These pro-
portions vary with species and with season. On the other
hand, -chitin is associated with a higher protein content but
lower carbonate concentration. Chitin is extracted by acid
treatment to dissolve the calcium carbonate followed by al-
kaline extraction to dissolve the proteins and by a depigmen-
tation step to obtain a colourless product mainly by remov-
ing the astaxantine [4].
2.2. Chitin Deacetylation
Chitosan is prepared by hydrolysis of acetamide groups
of chitin. This is normally conducted by severe alkaline hy-
drolysis treatment due to the resistance of such groups im-
posed by the trans arrangement of the C2-C3 substituents in
the sugar ring [5]. Thermal treatments of chitin under strong
aqueous alkali are usually needed to give partially deacety-
lated chitin (DA lower than 30%), regarded as chitosan.
Usually, sodium or potassium hydroxides are used at a con-
centration of 30-50% w/v at high temperature (100C).
In general, two major different methods of preparing chi-
tosan from chitin with varying degree of acetylation are
Table 1. Number of Scientific Publications Related to Chitin and Chitosan. Source: Scopus. Publication Year After 2000

Search Reviews Articles Patents
Chitin and not chitosan 182 2741 9064
Chitosan and not chitin 401 5959 20041
Chitin and chitosan 119 2040 11804
O
H
HO
H
HO
H
H
NH
H
OH
C
O
CH
3
O
H
O
H
HO
H
H
NH
H
OH
O
H
O
H
HO
H
H
NH
H
OH
OH
C
O CH
3
C
O
CH
3
n
O
H
HO
H
HO
H
H
NH
H
OH
C
O
CH
3
O
H
O
H
HO
H
H
NH
2
H
OH
O
H
O
H
HO
H
H
NH
2
H
OH
OH
n
A
B
Functional Characterization of Chitin and Chitosan Current Chemical Biology, 2009, Vol. 3, No. 2 205
known. These are the heterogeneous deacetylation of solid
chitin and the homogeneous deacetylation of pre-swollen
chitin under vacuum (by reducing pressure) in an aqueous
medium. Heterogeneous deacetylation, which is the pre-
ferred industrial treatment, involves preferential reaction in
the amorphous regions of the polymer, leaving almost intact
the intractable crystalline native regions in the parent chitin.
Alternatively, homogeneous modification is conducted by
use of moderately concentrated alkali (13% w/w) acting on
pre-swollen chitin to improve the interaction with the alkali
and left to react at 25-40C for 12-24 hours.
In both, heterogeneous or homogeneous conditions, the
deacetylation reaction involves the use of concentrated alkali
solutions and long processing times which can vary depend-
ing on the heterogeneous or homogeneous conditions from 1
to nearly 80 hours. Factors that affect the extent of deacetyla-
tion include concentration of the alkali, previous treatment,
particle size and density of chitin. The last two factors affect
penetration rate of the alkali into the amorphous region and
to some extent also into the crystalline regions of the poly-
mer, needed for the hydrolysis to take place. In practice, the
maximal DD that can be achieved in a single alkaline treat-
ment is about 75-85% [3]. In general, during deacetylation,
conditions must be the proper ones to deacetylate, in a rea-
sonable time, the chitin to yield a chitosan soluble in diluted
acetic acid.
Thiophenol and NaBH
4
have been used as oxygen scav-
enger and reducing agents, respectively, thus effectively re-
sulting in a product of greater viscosity [6]. Also, treatments
with concentrated NaOH in the presence of water-miscible
diluents such as 2-propanol, 2-methyl-2-propanol, polyethyl-
ene glycol dimethyl ether, acetone or paraffin oil have en-
abled the volume of concentrated NaOH required to be re-
duced by at least 85%. Several alternative processing meth-
ods have also been developed to reduce the long processing
times and large amounts of alkali typically needed to deace-
tylate chitin to an acid-soluble derivative. Examples of these
include the use of successive alkali treatments using thio-
phenol in DMSO [7]; thermo-mechanical processes using a
cascade reactor operated under low alkali concentration [8];
flash treatment under saturated steam [9]; use of microwave
dielectric heating [10]; and intermittent water washing [11].
There is evidence that in certain bacteria and fungi, en-
zymatic deacetylation can take place [12]. Deacetylases have
been isolated from various types of fungi, namely Mucor
rouxii, Aspergillus nidulans and Colletotrichum lindemuthi-
anium. However, the activity of these deacetylases is se-
verely limited by the insolubility of the chitin substrate.
There have been some attempts to use amorphous chitin of
high DA as a substrate for the deacetylase enzyme, however
no acid-soluble chitosan could be isolated and characterized
[13]. The lack of solubility of chitinous substrates with high
DA in aqueous solvents still represents a practical limitation
for the preparation of chitosan using the chitin deacetylase
system, a process which so far has been achieved in vivo
[14].
2.3. Chitosan Depolymerization
The main limitations in the use of chitosan in several
applications are its high viscosity and low solubility at neu-
tral pH. Low molecular weight (Mw) chitosans and oli-
gomers can be prepared by hydrolysis of the polymer chains.
For some specific applications, these smaller molecules have
been found to be much more useful. Chitosan depolymeriza-
tion can be carried out chemically, enzymatically or physi-
cally. Chemical depolymerization (Fig. 3) is mainly carried
out by acid hydrolysis using HCl or by oxidative reaction
using HNO
2
and H
2
O
2
[15]. It has been found to be specific
in the sense that HNO
2
attacks the amino group of D-units,
with subsequent cleavage of the adjacent glycosidic linkage.
In the case of enzymatic depolymerization, low molecular
weight chitosan with high water solubility were produced by
several enzymes such as chitinase, chitosanase, gluconase
and some proteases. Non-specific enzymes including
lysozyme, cellulase, lipase, amylase and pectinase that are
capable of depolymerizing chitosan are known [16]. In this
way, regioselective depolymerization under mild conditions
is allowed. Physical depolymerization yielding dimers, trim-
ers and tetramers has been carried out by radiation (Co-60
gamma rays) but low yields have been achieved [17].

Fig. (3). Chemical depolymerization of chitosan.
2.4. Influence of the Preparation Methods on the Physi-
cochemical Characteristics
The preparation method is a factor that affects the sample
characteristics. Early studies have clearly demonstrated that
specific characteristics of these products (Mw, DD) depend
on the process conditions.

Fig. (2). Three polymorphic configurations of Chitin (A) -chitin, (B) -chitin and (C) -Chitin.
O O
O
O
O
O
NHR'
HOH
2
C
NH
3
NHR'
OR
R =H, GlcN, GlcNAc
R' =H, Ac
Chitosan
O
NHR'
O
HO
HOH
2
C
NHR'
OR
HO
HNO
2
CHO
HO
HOH
2
C
RO
HO
HOH
2
C
O
HOH
2
C
HO
HO
HO
RO
HOH
2
C
Typically commercial chitins are prepared by a first step
of deproteinisation followed by a second step of deminerali-
zation. In these conditions a collapsed chitin, in which the
native structure of the chitin is lost, is extracted. On the other
hand, compacted chitin, in which the native chain and fi-
brous structures are intact and stabilized, is extracted when
demineralization occurred in the first step. Another way to
damage chitin structure was found to be even brief exposure
to bleaching agents [18].
The DA value of the bulk molecules depends directly on
the process conditions. Early studies by Kurita and co-
workers showed that chitosan produced under homogeneous
conditions presented broad X-ray diffraction patterns, which
was interpreted as a consequence of a more randomly dis-
tributed fine structure in terms of A and D groups [19,20]. It
has become evident that the overriding factor regarding the
fine structure of chitosan is the chemical polydispersion of
the DA value [21].
During chitosan deacetylation, the degradation of the
polymeric chain takes place. At the same time, the crystallin-
ity of chitosan can be damaged by using harsh reaction con-
ditions [22]. Taking these two facts into account, the reaction
conditions must be controlled when preparing chitosan [23].
Our findings have shown that the proper conditions to deace-
tylate chitin avoiding high degradation involve using hetero-
geneous conditions with NaOH 75% (w/v) and a temperature
of 110C [24]. The type of crustacean and the chitin isolation
process are also factors that affect chitosan quality [25].
3. METHODS OF CHARACTERIZATION
As will be shown in this review, chitin and chitosan char-
acteristics have a great effect on their properties and hence
on their possible applications. In fact, not every chitin or
chitosan sample can be used for the same applications. That
is why a complete characterization of the samples is manda-
tory.
Three crystalline forms are known for chitin: -, - and
-chitins. Chitosan is also crystalline and shows polymor-
phism depending on its physical state. Depending on the
origin of the polymer and its treatment during extraction
from raw resources, the residual crystallinity may vary con-
siderably. Crystallinity is maximal for both chitin (i.e. 0%
deacetylated) and fully deacetylated chitosan (i.e. 100%).
Rinaudo has reported in a recent review that the origin of
chitin influences not only its crystallinity and purity but also
its polymer chain arrangement, and hence its properties [26].
It has also been reported that the surface area of the material
is related to the source (i.e., crab>lobster >shrimp).
The main parameters affecting the polymer properties are
DD, Mw, polydispersity and crystallinity. For applications
related to human consumption such as food and medical ap-
plications, the purity (ash content), the moisture and the con-
tent of heavy metals, endotoxin and proteins must be deter-
mined.
It has been reported that the DD is one of the most im-
portant chemical characteristics, [27] which could influence
the performance of chitosan in many of its applications [28].
The influence of average Mw on the viscosity development
of aqueous solutions plays a significant role in the biochemi-
cal and biopharmacological significance of chitosan [29]. It
is important to note that due to its low solubility chitin Mw is
not easily determined.
As is shown in Table 2, various methods have been re-
ported for the determination of chitin and chitosan character-
istics [30-45]. Different results are obtained when using
methods based on different principles. Therefore, it is impor-
tant to indicate the characterization method. Today, even the
best characterized chitosans available in the market are usu-
ally described only with regard to their average degree of
acetylation and their average degree of polymerization (DP),
their ash content and the absence of contaminating bacteria,
in some cases also indicating the polydispersity index. In
addition to the above criteria, the distribution of the acetyl
groups along the linear backbone of the chitosan molecules
may be of crucial importance in defining the interactions
with the biological systems [46]. For further information
about preparation of chitin and chitosan, characterization and
Table 2. Physicochemical Characteristics of Chitin and Chitosan and the Determination Methods

Physicochemical Characteristics Determination Methods
DD Infrared spectroscopy [30,31,35]
First derivative UV-spectrophotometry [32, 33]
Nuclear magnetic resonance spectroscopy (
1
HNMR) and (
13
CNMR) [34-37]
Conductometric titration [37]
Potentiometric titration [38]
Differential scanning calorimetry [39]
Average Mw and/or Mw distribution Viscosimetry [40]
Gel Permeation chromatography [41]
Light scattering [42]
Crystallinity X-ray Diffraction [3, 43]
Moisture content Gravimetric analysis [44]
Ash content Gravimetric analysis [44]
Protein Bradford method [45]
DD: deacetylation degree.
chemistry Rinaudo and Kuritas reviews are recommended
[26,47].
4. REGULATORY ASPECTS
Chitosan has been approved as functional food in some
Asian countries (J apan, Korea) during the last decade. The
inclusion of chitin and chitosan was considered in 2003 by
the Codex Alimentarius Commission but it is not currently
listed in the General Standard for Food Additives nor has it
been authorized as a food ingredient in the EU. Although
several studies have shown that this compound is not toxic,
no long-term studies of human safety have been reported.
In the field of medical applications, chitosan has not been
approved yet by the FDA. However, The American Society
of Testing Materials (ASTM F04 division IV) is making a
concerned effort to establish standard guidelines for tissue-
engineered medical products (TEMPs). The F2103 guide
covers the evaluation of chitosan salts suitable for use in
medical applications considering aspects such as control of
protein content and, hence, potential for hypersensitivity,
endotoxin content, and total bioburden [44]. The F2260-03
guide covers the determination of DD while the WK965
guide covers the determination of Mw of chitosan and chito-
san salts [48,49].
A derivative of chitosan (chitosan hydrochloride) has
been included in the European Pharmacopoeia in 2002 [50].
This monograph includes tests for heavy metal as contami-
nats but bioburden, sterility and bacterial endotoxins are not
addressed. Taking into account that purity, which is quanti-
fied as the remaining ashes, proteins, insolubles and also the
bio-burden (microbes, yeasts and moulds, endotoxins,...), is
vital particularly for high value products, a more detailed
characterization is needed. Further information regarding this
topic is found in reference [51].
5. BIOLOGICAL PROPERTIES OF CHITIN AND
CHITOSAN
Chitin and chitosan are currently receiving a great deal of
interest as regards medical and pharmaceutical applications
because they have interesting properties that make them suit-
able for use in the biomedical field, such as biocompatibility,
biodegradability and non toxicity. Moreover, other properties
such as analgesic, antitumor, hemostatic, hypocholes-
terolemic, antimicrobian, and antioxidant properties have
also been reported [1,52,53].
A deeper understanding of the mechanism of these prop-
erties makes it necessary for chitosan to be well character-
ized and purified from accompanying compounds [54]. In
addition, chitins and chitosans derivatized in a variety of
fashions can be used to prove molecular hypothesis for the
biological activity. Since the majority of the biological prop-
erties are related to the cationic behaviour of chitosan, the
parameter with a higher effect is the DD. However, in some
cases, the Mw has a predominant role.
In addition to the DD and Mw, other properties such as
chain conformation, solubility or degree of substitution have
been considered. Chitosans produced by heterogenous deace-
tylation, with a block arrangement of acetylated and deacety-
lated units, have a tendency to form aggregates in aqueous
solutions [55]. Extensive aggregation and intermolecular
interactions may reduce available sites on the chitosan mole-
cule. This may account for some of the differences between
reported effects of chitosan, especially if the authors did not
pay close attention to the preparation of chitosan dispersions
or if the preparation procedure in these studies was different
[56]. The relationship between some chitin and chitosan bio-
logical properties and their physicochemical characteristics
are shown in Table 3.
Table 3. Relationship Between Chitin and Chitosan Biologi-
cal Properties and their Characteristics

Property Characteristic
Biodegradability DD, distribution of acetyl groups, Mw
Biocompatibility DD
Mucoadhesion DD, Mw (only chitosan)
Hemostatic DD, Mw
Analgesic DD
Adsorption enhancer DD (only Chitosan)
Antimicrobian Mw
Anticholesterolemic DD, Mw, viscosity
Antioxidant DD, Mw
Mw: molecular weight.
5.1. Biodegradability
Chitin and chitosan are absent from mammals but they
can be degraded in vivo by several proteases (lysozyme, pa-
pain, pepsin). Their biodegradation leads to the release of
non-toxic oligosaccharides of variable length which can be
subsequently incorporated to glycosaminoglycans and gly-
coproteins, to metabolic pathways or be excreted [57].
Lysozyme, a non-specific protease present in all mam-
malian tissues, seems to play a degradation role on chitin and
chitosan. The degradation kinetics seem to be inversely re-
lated to the degree of crystallinity which is controlled mainly
by the degree of deacetylation. Moreover, the distribution of
acetyl groups also affects biodegradability since the absence
of acetyl groups or their homogeneous distribution (random
rather than block) results in very low rates of enzymatic deg-
radation [2,58].
Finally, several studies reported that the length of the
chains (Mw) also affects the degradation rate [59-61]. The
understanding and control of the degradation rate of chitin
and chitosan-based devices is of great interest since degrada-
tion is essential in many small and large molecule release
applications and in functional tissue regeneration applica-
tions. Ideally, the rate of scaffold degradation should mirror
the rate of new tissue formation or be adequate for the con-
trolled release of bioactive molecules. Thus, it is important
to understand and control both the mechanism and the rate
by which each material is degraded.
The degradation rate also affects the biocompatibility
since very fast rates of degradation will produce an accumu-
lation of the amino sugars and produce an inflammatory re-
sponse. Chitosan samples with low DD induce an acute in-
flammatory response while chitosan samples with high DD
induce a minimal response due to the low degradation rate.
Degradation has been shown to increase as DD decreases
[62-64]. Kofuji et al. investigated the enzymatic behaviours
of various chitosans by observing changes in the viscosity of
chitosan solution in the presence of lysozyme [65]. They
found that chitosan with a low DD tended to be degraded
more rapidly. However, other authors reported that differ-
ences in degradation are due to variations in the distribution
of acetamide groups in the chitosan molecule [2,66]. This
occurs due to differences in deacetylation conditions which
influences viscosity of the chitosan solution by changing the
inter- or intra-molecular repulsion forces [64]. Therefore, It
can be concluded that it is impossible to estimate biodegra-
dation rate from the DD alone.
5.2. Biocompatibility
Both chitin and chitosan show very good compatibility
but this property depends on the characteristics of the sample
(natural source, method of preparation, Mw and DD). Due to
its higher versatility and biological properties the majority of
the assays have been carried out on chitosan samples.
Although the gastrointestinal enzymes can partially de-
grade both chitin and chitosan, when both polymers are
orally administered they are not absorbed. For this reason,
they are considered as not bioavailable. Chitosan shows a
LD
50
of around 16g/kg, very similar to the salt and glucose
values in assays carried out on mice [67]. Toxicity of chito-
san is reported to depend on DD. Schipper et al. reported
that chitosans with DD higher than 35% showed low toxic-
ity, while a DD under 35% caused dose dependant toxicity.
On the other hand, Mw of chitosan did not influence toxicity
[68] .
Chitosan presents higher cytocompatibility in vitro than
chitin. The cytocompatibility of chitosan has been proved in
vitro with myocardial, endothelial and epithellial cells, fi-
broblast, hepatocytes, condrocytes and keratinocytes [69].
This property seems to be related to the DD of the samples.
When the positive charge of the polymer increases, the inter-
actions between chitosan and the cells increase too, due to
the presence of free amino groups. The adhesion and prolif-
eration of keratinocytes and fibroblasts on several chitosan
films with different DDs depend on both, DD and cell type.
In both cells, the percentage of cell adhesion was strongly
dependent of the DD, increasing with this parameter. The
type of cell was a factor that also affected the adhesion, be-
ing more favourable for fibroblasts which exhibit a more
negative charge surface than for keratinocytes. On the other
hand, the proliferation decreased considerably by increasing
the DD.
Residual proteins in chitin and chitosan could cause al-
lergic reactions such as hypersensitivity. The protein content
in a sample depends on the source of the sample and, espe-
cially, on the method of preparation.
5.3. Haemostatic
It has been reported that chitosan, as well as sulphated
chitosan oligomers, presents anticoagulant activity tested in
vitro [70]. The anticoagulant activity of chitosan seems to be
related to its positive charge since red blood cells mem-
branes are negatively charged and chitin is less effective than
chitosan [71, 72]. Chitosan Mw also affects the binding or
agglutination of red blood cells [73]. In a recent paper, a
comparative study has been carried out among solid-state
chitosan and chitosan acetic acid physiological saline solu-
tion. Several chitosan samples with Mw from 2000 to 400
kDa and DD from 90 to 70% were tested. It was found that
solid-state chitosan and chitosan acetic acid physiological
saline solution followed different haemostatic mechanisms.
When blood was mixed with chitosan acetic acid physiologi-
cal saline solution, the erythrocytes aggregated and they
were deformed. The DD, especially a high DD, in the chito-
san acetic acid physiological saline solution, had a signifi-
cant effect on the unusual aggregation and deformation of
erythrocytes, compared with the effect of Mw within a range
between 10
5
and 10
6
. However, this phenomenon could not
be observed in solid-state chitosan soliquoid. Solid-state chi-
tosan with a high DD bound more platelets and was more
haemostatic [74].
5.4. Analgesic Effect
Several authors have reported that both chitin and chito-
san show analgesic effects [75-77]. Okamoto et al. have
studied the analgesic effect of chitin and chitosan on in-
flammatory pain due to intraperitoneal administration of
acetic acid and have proposed a mechanism for this analgesic
effect [78]. These authors found that chitosan showed a
greater effect than chitin. This difference was explained by
the different action mechanism of the two polymers. The
results suggested that the main analgesic effect of chitosan is
the absorption of proton ions released in the inflammatory
area.
Due to its polycationic nature, the free primary amino
groups of chitosan can protonate in the presence of proton
ions and the reduction in the pH is the main cause of the
analgesic effect. On the other hand, chitin was also able to
slightly absorb the proton ions but the concentration needed
to show the same effect as chitosan was lower than expected.
From experimental data, it was concluded that the analgesic
effect was due to the absorption of bradykinin, one of the
main substances related to pain.
5.5. Antitumor Activity
An antitumor activity of chitosan has been claimed by
inhibition of the growth of tumor cells mainly due to an im-
mune stimulation effect. However, this property is very con-
troversial [73].
J eon and Kim have found that chitosan oligomers possess
antitumor activities tested both in vitro and in vivo [79].
Studies carried out using mice that had ingested low-Mw
chitosan revealed significant antimetastatic effects of chito-
san against Lewis lung carcinoma. Partially deacetylated
chitin as well as chitin with a carboxymethyl group have also
been effective to demote tumor progression [80]. The sug-
gested mechanism involves immunostimulating effects of
chitin and its carboxymethyl derivatives via stimulation of
cytolytic T-lymphocytes. This activity increases with smaller
molecular sizes and it is suggested that they have immu-
nostimulating effects that activate peritoneal macrophages
and stimulate non-specific host resistance. However, higher
Mw oligomers have also exhibited antitumor activity. The
same mechanism has been suggested for their activity via
increased production of lymphokines by activated lympho-
cytes [81].
Ueno et al. studied the effect of chitosan on tumor
growth and metastasis. The activation of macrophages by
chitosan is suggested to mediate its antitumor effects in vivo,
while its angiogenic inducing properties may be the harmful
effects of chitosan, such as promotion of tumor growth and
invasion [82].
5.6. Mucoadhesion
Several factors affect chitosan mucoadhesion, such as
physiological variables and the physicochemical properties
of chitosan. The mucus is composed of a glycoprotein called
mucin, which is rich in negative charges since it has sialic
acid residues. In the stomach, chitosan is positively charged
due to the acidic environment and, therefore, it can interact
with mucin by electrostatic forces. The extent of this union
depends on the amount of sialic acid present in the mucin
and on the Mw and DD of chitosan. It has been found that
when the Mw of chitosan increases, the penetration in the
mucin layer also increases and hence the mucoadhesion is
stronger [83]. On the other hand, a higher DD leads to an
increase in charge density of the molecule and the adhesive
properties become more relevant [84].
Huang et al. evaluated the effects of Mw and DD on the
cellular uptake and in vitro cytotoxicity of chitosan mole-
cules and nanoparticles [59]. They found that the binding
affinity and uptake capacity of chitosan nanoparticles de-
creased when decreasing polymer Mw and degree of deace-
tylation. The effect of the degree of deacetylation was
greater than the effect of Mw because of its effect on the zeta
potential of the nanoparticles. However, the uptake of chito-
san molecules was less dependent on Mw and degree of
deacetylation.
El-Kamel et al. developed mucoadhesive micromatricial
chitosan/poly(-caprolactone) films for the treatment of
periodontal diseases [85]. These authors found that films
containing different Mw chitosans had different forces of
adhesion but statistical analysis revealed that there was no
significant difference in bioadhesion force between the films.
On the contrary, Roldo et al. showed that the maximal de-
tachment force of medium Mw chitosan was higher than that
of both low and high Mw chitosans [86].
5.7. Permeation Enhancing Effect
It has been reported that chitosan acts as a permeation
enhancer by opening epithelial tight junctions [87, 88]. The
mechanism underlying this behaviour is based on the interac-
tion of positively charged chitosan and the cell membrane
resulting in a reorganization of the tight junction-associated
proteins [89].
Schipper et al. investigated the effect of chitosan struc-
tural characteristics (Mw and DD) on their absorption en-
hancing properties in vitro (Caco-2 cell monolayers), using
chitosan hydrochloride salts at pH 5.5 [68]. It was found that
the capacity of chitosan to improve mannitol transport is
dependent on Mw and the DD; accordingly, while chitosans
with a high DD were efficient as permeation enhancers at
low and high Mw, those with low degrees of deacetylation
were efficient only at high Mws. Subsequently published
articles in this field agree that >80% deacetylation affords
the greatest promoter effect on cells in culture [89,90].
Soane et al. investigated the effect on mucociliary trans-
port velocity of five different types of chitosan with varying
Mws and degrees of deacetylation. The five types of chitosan
tested were shown to have no toxic effect on the frog palate
clearance mechanism [91]. The cilia beat frequency in
guinea pigs after nasal administration of chitosan solution
was also studied for 28 days and none of the chitosans used
showed any effect on the cilia frequency, which suggests that
using various types of chitosan for nasal delivery applica-
tions is not harmful.
5.8. Anticholesterolemic
There are several proposed mechanisms for cholesterol
reduction by chitosan. The latest findings in this field con-
sider more than one hypothesis. The entrapment caused by a
viscous polysaccharide solution is thought to reduce the ab-
sorption of fat and cholesterol in the diet. On the other hand,
the presence of the amino group in its structure determines
the electrostatic force between chitosan and anion sub-
stances, such as fatty acids and bile acids. Muzzarelli et al.
propose a spontaneous formation of insoluble chitosan salts
from bile acids whose hydrophobic nature should permit the
collection of cholesterol and lipids via hydrophobic interac-
tion [92]. A commercial food grade chitosan of DD 87 and
average Mw of 150 kDa was used to demonstrate this theory.
The interaction between chitosan and anionic surface-
active materials (phospholipids, bile acids) depends on its
three types of reactive functional groups: the amino group at
the C2 position and primary and secondary hydroxyl groups
at the C-3 and C-6 positions, respectively. Thongngam et al.
have demonstrated the formation of micelle-like clusters
within the chitosan structure in its interactions with a model
bile salt [93,94]. Another mechanism accounts for the ad-
sorption of chitosan to the surface of the emulsified lipid and
the formation of a protective coating that might prevent the
lipase/co-lipase from adsorbing to the droplet surfaces and
gaining access to the lipids inside the droplets [95].
Although great effort has been made to make a correla-
tion between the physicochemical characteristics of chitosan
and its fat-binding capacity, only some significant relation-
ships have been demonstrated. No et al. used six commer-
cially available chitosans with varying physicochemical
characteristics and showed that the fat binding capacity was
negatively correlated to the bulk density in a significant way
whereas it showed a trend to positively correlate with the
Mw [96]. The same group studied the fat binding capacity of
five chitosans of increasing Mw (range 500-800 kDa) pre-
pared by different depolymerization times, keeping a similar
DD, and found that the sample showing significant higher
activity was the one with the second lowest Mw [97].
In another study, a chitosan sample was submitted to
degradation with irradiation and sonolysis, and five decreas-
ing Mw where produced in the range 25-400 kDa. Samples
showed a trend to increase the fat-binding activity with de-
creasing Mw using a biopharmaceutical model of digestive
track [98]. Different experimental designs have been used
with the aim of mimicking the reactions taking place in the
stomach and duodenum. A digestive chemical model has
been used to study the interaction between chitosans of dif-
ferent viscosity and DD and sunflower oil. Although a nega-
tive correlation was found between the percentage of en-
trapped oil and increasing oil addition, no significant differ-
ences where found in chitosan behaviour according to its
characteristics [99]. Another in vitro human digestion model
was used to check the adsorption of chitosan to the fat drop-
lets. It was observed that the high Mw chitosan adsorbed to
the droplet more strongly than the low Mw. The reasons
proposed for this phenomenon were the different conforma-
tions of the chitosan molecule, with cationic loops and tails
in the high Mw and its higher surface activity [100].
A recent contribution has examined and compared eleven
chitosan preparations for their in vitro fat-binding capacity,
potency to bind individual bile acids, DD, solution viscosity,
and swelling volume. It was noted that the chitosan sample
having the strongest binding capacity against a selected bile
acid did not necessarily exhibit the strongest binding capac-
ity against other bile acids. No correlation was detected be-
tween individual bile acid-binding capacity and any other
tested physico-chemical properties of chitosan. These data
suggested that Mw, as reflected by solution viscosity, DD, or
swelling capacity might not be used to predict the bile acid-
binding capacity of chitosan [101].
5.9. Antimicrobial Activity
The antimicrobial activity of chitin, chitosan, and their
derivatives against different groups of microorganisms, such
as bacteria, yeast, and fungi, has received considerable atten-
tion in recent years. Two main mechanisms have been sug-
gested as the cause of the inhibition of microbial cells by
chitosan. The interaction with anionic groups on the cell sur-
face, due to its polycationic nature, causes the formation of
an impermeable layer around the cell, which prevents the
transport of essential solutes. It has been demonstrated by
electron microscopy that the site of action is the outer mem-
brane of gram negative bacteria. The permeabilizing effect
has been observed at slightly acidic conditions in which chi-
tosan is protonated, but this permeabilizing effect of chitosan
is reversible [102].
The second mechanism involves the inhibition of the
RNA and protein synthesis by permeation into the cell nu-
cleus. Liu et al. have observed labelled chitosan oligomers
with Mw from 8 to 5 kDa inside the E. coli cell and they
showed good antibacterial activities [103]. In this case the
Mw is the decisive property (Table 4).
Table 4. Influence of Chitosan DD and Mw on Antimicrobial
Activity

Physico-Chemical Property Effect on Antimicrobial Activity
electrostatic binding to mem-
brane
DD
permeabilizing effect
Mw permeation into the cell nucleus
Other mechanisms have also been proposed. Chitosan
may inhibit microbial growth by acting as a chelating agent
rendering metals, trace elements or essential nutrients un-
available for the organism to grow at the normal rate. Chito-
san is also able to interact with flocculate proteins, but this
action is highly pH-dependent [104]. Several authors have
proposed that the antimicrobial action of chitosan against
filamentous fungi could be explained by a more direct dis-
turbance of membrane function [105]. However, it is not
clear whether the antimicrobial activity of chitosan is caused
by growth inhibition or cell death.
Antibacterial activities were found to increase in the or-
der of N,O-CM-chitosan, chitosan, and O-CM-chitosan. The
first product, where amino and hydroxyl groups have been
substituted by carboxymethyl groups, contains fewer amino
residues. In the case of O-CM-chitosan, its number of amino
groups is not changed. Moreover, its carboxyl group may
have reacted with the amino groups intra- or intermolecu-
larly and charged these groups. The authors concluded that
the antibacterial activities of chitosan and carboxymethylated
derivatives depend on the effective number of NH
3
+
groups
[103].
Several studies prove that an increase in the positive
charge of chitosan makes it bind to bacterial cell walls more
strongly [106]. The relationship between Mw, number of
charges and antimicrobial activity has been pointed out by
Kim et al. [107]. They showed that O-CM chitosan derived
from degraded chitosan was more effective than plain chito-
san. This was attributed to the interaction of the COOH
group with the NH
2
group intra-or intermolecularly to impart
a charge, the number of NH
3
groups becoming larger. In the
case of native chitosan, an excessive concentration of amino
groups on O-CM chitosan promotes a structure that involves
cross-linking through strong intramolecular hydrogen bond-
ing, where the number of amino groups that are available to
attach bacterial surfaces is reduced.
In contrast, some authors have not found a clear relation-
ship between the degree of deacetylation and antimicrobial
activity. These authors suggest that the antimicrobial activity
of chitosan is dependent on both the chitosan and the micro-
organism used [108, 109]. Park et al. studied the antimicro-
bial activity of hetero-chitosans and hetero-COs with differ-
ent degrees of deacetylation and Mws against three Gram-
negative bacteria and five Gram-positive bacteria and found
that the 75% deacetylated chitosan showed more effective
antimicrobial activity compared with that of 90% and 50%
deacetylated chitosan [110].
5.10. Antioxidative Activity
Chitosan has shown a significant scavenging capacity
against different radical species, the results being compara-
ble to those obtained with commercial antioxidants. Samples
prepared from crab shell chitin with DD of 90, 75 and 50%
where evaluated on the basis of their abilities to scavenge
1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydroxyl
radical, superoxide radical and alkyl radical. The results re-
vealed that chitosan with higher DD exhibited the highest
scavenging activity [111].
On the other hand, chitosans of different size as well as
their sulphate derivatives were assayed against superoxide
and hydroxyl radicals. A negative correlation was found be-
tween chitosan Mw and activity (Table 5). The chitosan sul-
phated derivatives presented a stronger scavenging effect on
peroxide radicals but the chitosan of lowest Mw showed
more considerable ferrous ion-chelating potency than others
[112]. The chelation of metal ions is one of the reasons why
chitosan may be considered as a potential natural antioxidant
for stabilizing lipid containing foods to prolong shelf life.
Chitosans may retard lipid oxidation by chelating ferrous
ions present in the system, thus eliminating their prooxidant
activity or their conversion to ferric ion [113].
Table 5. Influence of Chitosan DD and Mw on Antioxidative
Activity

Physico-Chemical Property Effect on Antioxidant Activity
DD scavenging effect
Mw radical scavenging effect
ion-chelating potency
This activity has been also studied in chitooligosaccha-
rides (COS). Chitobiose and chitotriose have proved to be
more potent than three reference compounds (aminogua-
nidine, pyridoxamine and trolox) in scavenging hydroxyl
radicals while glucosamine and the corresponding N-
acetylchito-oligosaccharides did not show any capacity
[114]. Electron spin resonance spectrometry has been used to
follow the scavenging activity of chitooligosaccharide mix-
tures fractionated by ultrafiltration. This activity was shown
to be dependent on the Mw, the fraction 1-3 kDa having the
highest radical scavenging effect [115]. When the DD was
considered, a correlation between scavenging activity over
all tested free radicals with the increment of deacetylation
values of COS was found. Therefore, it has been pointed out
that the free amino groups in the hetero COS play an impor-
tant role in free radical scavenging activity, probably by
forming stable macromolecule radicals [116]. This capacity
of oligosaccharides has been further assayed in vivo. Yang et
al. assayed two different Mw COS (1.1 and 0.5 kDa) against
H
2
0
2
released from polymorphonuclear leukocytes stimu-
lated by phorbol-12-myristate-13-acetate in rats [117]. They
found that the radical scavenging capacity was higher for the
first COS.
6. BIOMEDICAL APPLICATIONS OF CHITIN AND
CHITOSAN
Due to its high biocompatibility, chitosan has been em-
ployed in drug delivery systems, implantable and injectable
systems such as orthopaedic and periodontal composites,
wound healing management and scaffolds for tissue regen-
eration [118,119].
6.1. Wound Healing
Chitin and chitosan activate immunocytes and inflamma-
tory cells such as PMN, macrophage, fibroblasts and angio-
endothelial cells. These effects are related to the DD of the
samples, chitin presenting a weaker effect than chitosan [82].
Chitosan oligomers have also exhibited wound-healing
properties, it is suggested that their wound-healing properties
are due to their ability to stimulate fibroblast production by
affecting the fibroblast growth factor. Subsequent collagen
production further facilitates the formation of connective
tissue [120].
Recently, the effects of chitin and chitosan oligomers and
monomers on wound healing have been studied [121]. This
study shows that in addition to chitin and chitosan, their oli-
gomers and monomers enhance wound healing acceleration.
Wound break strength and collagenase activity of the chito-
san group (D-glucosamine (GlcN), chito-oligosaccharide
(COS), chitosan) were higher than the chitin group (N-
acetyl-D-glucosamine (GlcNAc), chiti-oligosaccharide (NA-
COS), chitin). Collagen fibres run perpendicular to the inci-
sional line in the oligosaccharide group (NACOS, COS) and
many activated fibroblasts were observed in the histological
studies around the wound in the chitosan groups. The break
strength was stronger and more activated fibroblasts were
observed at higher DD.
The potential use of chitin oligosaccharides (DP2, DP3,
DP4, DP5 and DP7) in wound healing as well as their capac-
ity against chronic bowel disease has been studied. For the
first time, a mucin-stimulating effect of chitin oligomers
DP3 and DP5 has been observed in an ex-vivo model [122].
The wound healing effect of chitin and chitosan oligomers
and monomers is of great interest because in vivo lysozyme
degrades chitin and chitosan to these smaller molecules.
6.2. Drug Delivery Systems
An important application of chitosans in industry is the
development of drug delivery systems such as nanoparticles,
hydrogels, microspheres, films and tablets (Fig. 4). As a re-
sult of its cationic character, chitosan is able to react with
polyanions giving rise to polyelectrolyte complexes [123-
124]. Pharmaceutical applications include nasal, ocular, oral,
parenteral and transdermal drug delivery. Three main charac-
teristics of chitosan to be considered are: Mw, degree of ace-
tylation and purity. When chitosan chains become shorter
(low Mw chitosan), it can be dissolved directly in water,
which is particularly useful for specific applications in bio-
medical or cosmetic fields, when pH should stay at around
7.0.
In drug delivery, the selection of an ideal type of chitosan
with certain characteristics is useful for developing sustained
drug delivery systems, prolonging the duration of drug activ-
ity, improving therapeutic efficiency and reducing side ef-
fects. Kofuji et al. suggested that the physicochemical char-
acteristics of chitosan are important for the selection of the
appropriate chitosan as a material for drug delivery vehicles
[65]. Investigations have indicated that DD and Mw of chito-
san have significantly affected the role of chitosan in thera-
peutic and intelligent drug delivery systems [125, 126].
Mi et al. studied the gelation properties of microspheres
cross-linked with glutaraldehyde as it had significant effect
on drug incorporation [127]. Microspheres prepared with a
high Mw chitosan gelled faster than those prepared with a
low Mw because they have different activation energies of
gelation. Chitosan with short chains have higher activation
energy and need more time to interact with the other chains
and to gelate with glutaraldehyde.
Gupta and J abrail studied the effect of degree of deacety-
lation and cross-linking on physical characteristics, swelling
and release behaviour of centchroman loaded chitosan mi-
crospheres [128]. The DD controls the degree of crystallinity
and hydrophobicity in chitosan due to variations in hydro-
phobic interactions which control the loading and release
characteristics of chitosan matrices. The DD also controls
the degree of cross-linking of chitosan in the presence of any
suitable cross-linker. The higher the DD is, the higher the
number of free amino groups and therefore the degree of
covalent cross-linking increases [129]. When analyzing the
influence of cross-linking degree and degree of deacetylation
on size and morphology of the microspheres, these authors
reported that the size and the surface roughness decreased on
increasing the degree of cross-linking and the degree of
deacetylation. Zhang et al. also reported that a high degree of
chitosan deacetylation and narrow polymer Mw distribution
were shown to be critical for the control of particle size dis-
tribution [130].
A higher degree of cross-linking and a higher DD in chi-
tosan increase the compactness of matrices and its hydro-
phobicity, thus controlling the degree of swelling and diffu-
sivity of the drug entrapped in chitosan matrixes. It was ob-
served that a DD between 48-62% promotes maximal load-
ing capacity, due to the size of the cross-links and pores
formed. Regarding the release properties, a very low DD can
induce burst release [128].
In another study with chitosan microspheres loaded with
centchroman and crosslinked with glutaraldehyde, Gupta and
J abrail observed that the lower Mw of chitosan employed,
the lower sphericity of the microspheres obtained and these
microspheres were larger in size than those prepared with
medium-high Mw chitosan due to the poorer molecular
packing and crosslinking [131]. These results are in agree-
ment with those presented by Desai and Park, who studied
the influence of Mw of chitosan on chitosan-TPP micro-
spheres prepared by spray-drying [132]. They observed that
an increase of Mw also produced more spherical micro-
spheres, with greater size homogeneity and a smoother sur-
face. In addition they found that an increase in molecular
weight gave bigger microspheres as a result. Gupta and
J abrail in their study also found that microspheres prepared
with high Mw chitosan presented a very low degree of swel-
ling and a high degree of crosslinking, thus, those micro-
spheres prepared with medium Mw chitosan that lead to less
strong intermolecular interactions being more appropriate for
sustained release [131]. These results were also in agreement
with Desai and Park who observed that the release rate of
vitamin C was much lower as the Mw of chitosan used for
preparing microspheres increased [132]. They studied the
release kinetics and found that it followed Ficks law of dif-
fusion.
Low Mw chitosan leads to poor retention of centchroman
in microspheres due to a high degree of swelling and a frag-
ile network structure. The microspheres with medium Mw
chitosan showed an optimum loading efficiency [131]. Mi-
crospheres with medium Mw chitosan are more efficient in
releasing the centchroman in a controlled manner in com-
parison to low and high Mw chitosan microspheres. The ini-
tial burst release of centchroman from microspheres with
different Mws and different degrees of deacetylation of chi-
tosan varied linearly with the square root of the release time
indicating a diffusion-controlled release of centchroman
from these microspheres (n =0.5). However, the release of
centchroman in the controlled stage of drug release was
anomalous [133]. The initial slope of these curves was used
to calculate the diffusion coefficient (D) for centchroman
from chitosan microspheres. The value of the diffusion coef-
ficient for centchroman from microspheres decreased on
increasing the Mw of chitosan, and decreased on increasing
the DD in chitosan. This clearly indicates that the release of
centchroman from these microspheres is diffusion controlled
and the variation in the diffusion coefficient (D) of
centchroman on varying the Mw and degree of deacetylation
in chitosan is due to the variations in the structure of micro-
spheres [131]. The influence of chitosan DD and Mw on the
microspheres properties prepared as matrix for drug delivery
is shown in Table 6.
Desai and Park in the study of the influence of chitosan
Mw on chitosan-TPP microspheres found that it does not
affect the spray drying yield [132]. However, it has influence
on some parameters of the microspheres that have already
been commented on. In addition they studied the influence
on zeta potential and observed some differences that were
not very significant.
Chiou et al. investigated the effect of post-coating PLLA
microspheres with different chitosans on the initial burst and
controlling the drug release of the microspheres [134]. With-
out chitosan, 20% lidocaine was released within the first
hour and the time of 50% release was 25 hours. This period
was extended to 90 hours after coating with chitosan. They
observed that when applying chitosan of the same Mw, the

Fig. (4). (A) High Mw Chitosan (640 kDa) microspheres crosslinked with 0.2% TPP obtained by spray-drying. (B) Detail of the micro-
spheres.
efficacy of reducing the initial burst of drug release was
higher for a lower degree of deacetylation. With chitosan in
acetic acid solution, coating the microspheres with high Mw
and high viscosity could most effectively reduce the initial
burst and control drug release of PLLA microspheres. The
study indicated that manipulating the viscosity of the chito-
san solution was the most important factor in contributing to
controlling the drug release of chitosan post-coated PLLA
microspheres.
Table 6. Influence of Chitosan DD and Mw on Microspheres
Properties

Physico-Chemical Property Effect on Microsphere Properties
covalent crosslinking
size
surface roughness
swelling
compactness and hydrophobicity
loading capacity
DD
burst release
sphericity
morphology homogeneity
crosslinking
swelling
release rate
Mw
diffusion coefficient (D)
Kofuji et al. studied the relationship between physico-
chemical characteristics and functional properties of chitosan
such as the ability to form spherical gel, control of drug re-
lease from chitosan gel and biodegradation of chitosan [65].
They found that the formation of spherical chitosan gels in
aqueous amino acid solution or aqueous solution containing
metal ions was affected mainly by viscosity of the chitosan
solution. High concentration of chitosan species with a high
Mw could not be used to prepare chitosan spherical gel due
to its high viscosity and the use of very low concentration of
chitosan did not result in instantaneous spherical gel forma-
tion because the diffusion of chitosan within the preparative
medium was too rapid. The degree of deacetylation also had
an effect on spherical gel formation in the case of gelation of
chitosan by chelation with metal ions. Chitosan with high
degree of deacetylation was able to form spherical gel by
chelation due to higher availability of amino groups that
chelated with metal ions better than chitosan of low DD.
Only in the case of chelation with metal ions was the extent
of deacetylation related to drug release.
El-Kamel et al. developed mucoadhesive micromatricial
chitosan/poly(-caprolactone) films for the treatment of
periodontal diseases [85]. They examined the effect of dif-
ferent molar masses of chitosan on morphology of micropar-
ticles trapped in the films, water absorption, in vitro bioad-
hesion, mechanical properties and in vitro drug release. The
mean size of entrapped caprolactone particles was higher in
films containing higher Mw chitosan. These authors attrib-
uted this to the increased viscosity of the chitosan solution as
the Mw increased. After studying water absorption capacity,
results revealed that there was no statistically significant
difference in percentage water uptake with different Mw
chitosans. This result was in agreement with Roldo et al.
[86], who found no correlation between the Mw of chitosan
and its swelling behaviour.
The mechanical properties of films with different Mw
chitosans were also measured by El- Kamel et al. The tensile
strength (TS), the percentage elongation at break (% EB) and
the elastic modulus (EM) are important parameters to indi-
cate the strength and elasticity of the film [85]. They found
that medium Mw chitosan films had highest values for TS
and EM, followed by high Mw and low Mw chitosan films.
On the other hand, the highest % EB was obtained for low
Mw chitosan films, followed by high and medium Mw chito-
san films.
With regard to in vitro release studies, they found that the
amount of drug released from prepared films was similar for
films that contained low and medium Mw chitosan and
lower for the ones prepared with high Mw chitosan. This
behaviour was predictable, taking into account the direct
relationship between the molar mass of chitosan and the vis-
cosity of its solution. By increasing the viscosity of the
polymer, the diffusion of the drug through the formed gel
layer into the release medium was retarded [135]. The high
polymer viscosity may also affect the size of particles
formed by reducing the homogenization efficiency, leading
to the formation of larger PCL microparticles, as indicated
by the particle-size analysis studies. Therefore, the exposed
surface area is reduced and the release of the entrapped drug
is decreased.
6.3. Gene Delivery
Due to its positive charge, chitosan has the ability to in-
teract with negative molecules such as DNA. This property
was used for the first time to prepare a non-viral vector for a
gene delivery system by Mumper in 1995 [136]. The use of
chitosan as non-viral vector for gene delivery offers several
advantages compared to viral vectors. Mainly, chitosan does
not produce endogenous recombination, oncogenic effects or
immunological reactions [137]. Moreover, chitosan/pDNA
complexes can be easily prepared at low cost.
The Mw of chitosan is a key parameter in the preparation
of chitosan/pDNA complexes since transfection efficiency
correlates strongly with chitosan Mw. High molecular
weight chitosan renders very stable complexes but the trans-
fection efficiency is very low. To improve transfection effi-
ciency, recent studies have examined the use of low Mw
chitosans [138-146] and oligomers [147-149] in gene deliv-
ery vectors. It appears that a fine balance must be achieved
between extracellular DNA protection (better with high Mw)
versus efficient intracellular unpackaging (better with low
Mw) in order to obtain high levels of transfection. Lavertu et
al. studied several combinations of Mw and DA of chitosan
finding two combinations of high transfection efficiency
using a chitosan of 10 kDa and DD of 92 and 80%, respec-
tively [150].
Kiang et al. studied the effect of the degree of chitosan
deacetylation on the efficiency of gene transfection in chito-
san-DNA nanoparticles [151]. Highly deacetylated chitosan
(above 80%) releases DNA very slowly. They suggest that
the use of chitosan with a DD below 80% may facilitate the
release of DNA since it lowers the charge density, may in-
crease steric hindrance in complexing with DNA, and is
known to accelerate degradation rate. They reported an in-
crease in luciferase expression when the degree of deacetyla-
tion was decreased from 90% to 70%. Formulations with
62% and 70% deacetylation led to luciferase transgenic ex-
pression two orders of magnitude higher than chitosan with
90% deacetylation.
6.4. Tissue Engineering
Recent studies in regenerative tissue engineering suggest
the use of scaffolds to support and organize damaged tissue
because three-dimensional matrices provide a more favour-
able ambient for cellular behaviour. Due to their low immu-
nogenic activity, controlled biodegradability and porous
structure, chitosan scaffolds are promising materials for the
design of tissue engineered systems [152-154].
Tl et al. studied the influence of DD on some struc-
tural and biological properties of chitosan scaffolds for cell
culture and tissue engineering [155]. They observed that
chitosan scaffolds with low DD (75-85%) displayed a more
regular structure and the pores were fairly uniform and paral-
lel with a polygonal cross section. The lateral pore connec-
tivity was much lower than for scaffolds with high deacety-
lation degrees (>85%). It is known that the microstructure
such as pore size, shape and distribution, has prominent in-
fluence on cell intrusion, proliferation and function in tissue
engineering. Swelling studies were also performed but no
relationship was found between DD and swelling ratio. Me-
chanical testing of chitosan scaffolds showed that mechani-
cal strength was higher with higher DD. Biodegradability of
the scaffolds also depends on the DD. Cell attachment stud-
ies on the scaffolds showed that higher DD favoured cell
adhesion.
Other authors also reported that a lower degree of acety-
lation favoured cell adhesion [69,156]. The viability of fi-
broblasts on chitosan scaffolds with different DD was evalu-
ated. A significant increase in cell number was observed on
>85% deacetylated chitosan scaffolds. A high proliferation
trend was suggested when compared to low deacetylated
chitosan scaffolds.
Chitin and chitosan tubes for nerve regeneration were
prepared by Freier et al. [157]. The compressive strength of
these tubes was found to increase with decreasing acetyla-
tion. Both chitin and chitosan support adhesion and differen-
tiation of primary chick dorsal root ganglion neurons in vi-
tro, with significantly enhanced neurite outgrowth on chito-
san than on chitin films. The effect of DA on the cell adhe-
sion and biodegradation of chitin and chitosan films was
studied to find the most suitable conditions for cell compati-
bility and optimum biodegradation [158].
Injectable thermosetting chitosan hydrogels are attractive
systems for drug delivery and tissue engineering that com-
bine biodegradability, biocompatibility and the ability to
form in situ gel-like implants. Thermally-induced gelation
relies advantageously on biopolymer secondary interactions,
avoiding potentially toxic polymerization reactions that may
occur with in situ polymerizing formulations. Besides -
glycerophosphate [159], other molecules such as 1,3-
propanediol, 1,2-propanediol as well as glycerol, mannitol or
polyoses such as trehalose have been reported to induce the
thermogelation of chitosan [160].
Schuetz et al. studied the effect of the Mw of chitosan on
the properties of the thermosetting chitosan hydrogels during
storage and sterilization by autoclaving [160]. The autoclav-
ing process produced a reduction of the Mw of the chitosan
samples which was affected by the initial Mw of the sample.
The authors concluded that chitosans exhibiting highly re-
duced Mw when autoclaved might not be adapted to this
sterilization method in specific applications where maximal
mechanical performance is essential for implant function.
With regard to the freeze storage, low Mw chitosan ther-
mogels or those prepared with low enough concentration
might be kept frozen for prolonged storage.
Porous scaffolds were prepared by freeze-drying a solu-
tion of collagen and chitosan, followed by cross-linking by
dehydrothermal treatment. The effect of the chitosan Mw
and the blending ratio was studied. The lysozyme biodegra-
dation test demonstrated that the presence of chitosan, espe-
cially the high-molecular-weight species, could significantly
prolong the biodegradation of collagen/chitosan scaffolds. In
vitro culture of L929 mouse connective tissue fibroblast evi-
denced that low-molecular-weight chitosan was more effec-
tive for promoting and accelerating cell proliferation, par-
ticularly for scaffolds containing 30% (w/w) chitosan. The
results elucidated that the blends of collagen with low-Mw
chitosan have a high potential to be applied as new materials
for skin-tissue engineering [161].
Apart from the aforementioned characteristics, which are
specific for each application, there is a degree of consensus
regarding general characteristics that must be present in chi-
tosan samples to be used in the field of biomedical applica-
tions (Table 7) [162, 163].
7. FOOD APPLICATIONS OF CHITOSAN
Chitosan offers a wide range of unique applications in the
food industry, including preservation of foods from micro-
bial deterioration, formation of biodegradable films, and
recovery of material from food processing discards. Moreo-
ver, it can act as a dietary fibre and as a functional food in-
gredient.
7.1. Dietary Ingredient
Chitosan has been used in multiple nutritional supple-
ment products due to its ability to bind fat. The in vivo stud-
ies are intended to demonstrate a significant reduction in the
body weight gain or the plasma lipid content of humans or
animals.
Recently, Liu et al. have reported that rats fed diets con-
taining the highest deacetylated chitosan significantly low-
ered plasma cholesterol and LDL-C, and increased HDL-C
level [164]. Chitosan with high Mw limited the body weight
gain of adult rats significantly. When the DD and particle
size were considered, chitosan with higher Mw also exhib-
ited better cholesterol-binding capacity in vitro. These results
indicated that the viscosity in the upper gastrointestinal tract
was not the major factor influencing the hypocholesterolae-
mic effect of chitosan. Nonetheless, they concluded that
when the particle is finer, and DD and Mw are relatively
high, the effect is better.
Zeng et al. studied the in vivo absorption phenomena of
different Mw chitosan in mice and found that absorption of
chitosan increased with the decrease of Mw and the increase
of watersolubility [165]. Chitosan with very high Mw was
very difficult to absorb and enter the blood. Chitooligomers
were easily degraded into much smaller molecules, quickly
absorbed and distributed to other places.
Sumiyoshi and Kimura examined the effects of various
water-soluble low Mw chitosans (average Mw: 21, 46 and
130 kDa) on pancreatic lipase activity, the 46 kDa chitosan
being the most effective in the inhibition of this enzyme
[166]. This chitosan prevented increases in body weight;
various white adipose tissue weights and liver lipids (choles-
terol and triacylglycerol) in mice fed a high fat diet, and fur-
ther increased the faecal bile acid and fat. This group had
previously reported that water- insoluble, high Mw chitosan
(650 kDa), which is the minimal size of that approved by the
J apanese Ministry of Health, Labour and Welfare as func-
tional food, prevented the increases in bodyweight and white
adipose tissue weights, hyperlipidaemia and fatty liver in-
duced by feeding the high-fat diet for 9 weeks, by inhibiting
the intestinal absorption of dietary fat [167].
The effect of differences in the viscosity of chitosan
preparations on plasma lipoprotein cholesterol and the lipid
peroxidation status in rats has been studied. The serum cho-
lesterol-lowering action of chitosan was reported to be inde-
pendent of its viscosity. However, a comparison of the liver
lipid-lowering and lipid oxidation effects of chitosan sam-
ples with different viscosity showed that the total liver lipid
and cholesterol-lowering action of chitosan was greater for
the high-viscosity samples when the DD of the preparations
were comparable [168].
The effects of chitosan properties on fat binding and fat
metabolism are shown in Table 8.
7.2. Food Preservative
Chitosans have been identified as versatile biopolymers
of natural origin for food preservation due to their antimi-
crobial action against food spoilage microorganisms and
antioxidant properties. The pH-dependent solubility allows
them to be formed into various shapes (beads, films and
membranes) using aqueous processing [169].
The results of the experiments indicate that, in general,
low (5-27 kDa) and medium (48-78 kDa) Mw chitosans and
high DD 85-98% effectively suppress the growth of both
gram-positive and gram-negative bacteria [106,170]. A study
of chitosan obtained from cuticles of housefly larvae points
to the fact that the antibacterial effect of chitosan decreases
with increase in Mw; in this case chitosans with Mw ranging
from 21 to 44 kDa were more effective than chitosans of 8
and 476 kDa [171].
However, very often the most effective Mw of chitosan
varies with the microorganism tested. In the case of Candida
kruisei, chitosan apparently cannot bind to the surface of the
cell wall of the fungus and penetrate inside. However, this
effect is apparently species-specific, because another Can-
dida species, C. albicans, was highly sensitive to all chito-
sans tested [106]. Liu et al. showed that at the high (200, 500
and 1000 ppm) and low (20 ppm) concentrations, the anti-
bacterial activity of chitosan had no relationship to the Mw.
However, at the middle concentration from 50 to 100 ppm,
with the decrease of Mw, antibacterial activities increased
[172].
Higher DD are related with better results. Tsai et al.
compared the antimicrobial activities of chitin and chitosan
obtained by chemical and biological treatments of shrimp
shell. The MICs, which were in the range of 50-200 ppm,
became smaller with increasing DD [56].
7.2.1. Food Emulsions
The antimicrobial properties of chitosan in a liquid me-
dium will be poorly represented in complex food systems
where the interaction of chitosans with other components
may modulate their activity [109]. Chitosan solubility in
aqueous acetic acid and its location at the interface are excel-
lent predispositions for its application as antimicrobial agent
in food emulsions [173]. Despite the fact that emulsions con-
tain large concentrations of oil that do not support growth,
these emulsions may contain spilage and pathogenic micro-
Table 7. Characterization of Chitosan for Medical Application [162, 163]

Variable Under Study Appropiate Characteristic
Moisture Content <10%
Ash content <0,2%
Protein content <0,3%
Insolubility <0,1%
Turbidity Cs 1%w/v in AcOH 1%v/v <15 NTU
Viscosity <5 cps
DD 70-100%
Heavy metal content As<10ppm, Pb<10ppm
Bioburden, aerobic bacteria plus fungi (<100CFU; abscence of pathogens)
Organoleptic properties No taste, no smell
Cs: chitosan.
NTU: Nefelometric turbidity unit.
CFU: Colony forming units.
organisms in the non-lipid phase. Mayonnaise has been
choosen as a model system where three target microorgan-
isms have been inoculated. The most effective Mw of chito-
san had been shown to vary with the microorganism tested.
Viable cell counts decreased significantly without chitosans,
although its addition markedly reduced the viable cell counts
as compared with those of controls [109].
Studies of the effect of solubility of chitosan revealed
that the water insoluble chitosan exhibited the antimicrobial
effect, whereas water-soluble chitosan itself had no signifi-
cant antimicrobial effect against both bacteria and yeast
[170]. However, Chung et al. have reported the metal-ion
chelating capacity and antibacterial activity of a chitosan-
glucosamine derivative prepared by the Maillard reaction.
This derivative appeared to be more effective than other chi-
tosans or chitosans derivatives as a natural bactericidal agent
[174].
The Maillard reaction has been used to develop biofunc-
tional biopolymers as food preservatives with broad antimi-
crobial effects. Chitosans of different degrees of polymeriza-
tion were mixed with lysozyme [175] and gluten peptides
[176] and conjugated through this reaction. The results dem-
onstrate that high Mw chitosan conjugates were very effec-
tive in improving the bactericidal activity of proteins or pep-
tides compared to low Mw chitosan conjugates. It has been
shown that the Maillard reaction can be successfully em-
ployed to generate products from -lactoglobulin and chito-
san, which exhibit improved bactericidal properties with
respect to -lactoglobulin alone [177].
7.2.2. Aqueous Systems
Apple juice has been used as an aqueous model system to
study the antioxidative activity of chitosans with different
Mws. Low Mw chitosan exhibited stronger scavenging activ-
ity than medium or high Mw and ascorbic acid, which was
used as a positive scavenger. However the authors conclude
that in vivo antioxidant activity and the various antioxidant
mechanisms must be further investigated [178].
7.2.3. Solid Matrix Systems
The iron bound to fish tissue proteins such as myoglobin,
haemoglobin, ferritin and transferrin may be released during
storage and cooking, thus activating oxygen and initiating
lipid oxidation. Kim and Thomas have examined chitosans
of different Mw as antioxidative agents in salmon based on
the measurement of 2-thiobarbituric acid-reactive substances
(TBARS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scav-
enging activity [179]. The 30 kDa chitosan showed the high-
est scavenging activity compared to 90 and 120 kDa chito-
san. The increase in concentration of 30kDa chitosan re-
sulted in the increase of total amino groups responsible for
scavenging more radicals.
Fatty (herring) and lean (cod) fishes have been used as
model systems to assay the antioxidant activity of 3 chito-
sans prepared with different deacetylation times of the same
sample. The lowest viscosity chitosan presented the highest
antioxidant effect. This was attributed to the lower chelation
by high viscosity (high Mw) chitosan, as the intramolecular
electric repulsive forces would increase the hydrodynamic
volume by extended chain conformation. However, the DD
was pointed to as another factor that may be involved in the
chelation ability of chitosans [108-181].
7.2.4. Edible Films and Coatings
Coatings can retard ripening and water loss and reduce
decay but they may also alter the flavour. Semi-permeable
coatings such as chitosan may create a modified atmosphere
similar to the controlled atmosphere used in storage, but at a
lower cost [182]. Although many studies on chitosan coating
have been published, very few of them consider the influ-
ence of the physicochemical properties on their activity.
Shortfin squid chitosan and shrimp chitosan membranes
were tested for water vapour apparent permeability, swelling
in water, and mechanical properties, in order to evaluate the
effect of the acetylation degree and Mw of chitosan on these
properties. The results indicated that decreasing the number
of the bulky acetyl groups led to more intermolecular inter-
actions among polymer chains, namely hydrogen-bonding,
resulting in a tightening of the polymer network. Therefore,
lower water vapour permeability and swelling in water are
found, and the mechanical properties are improved.
Decreasing the molecular mass of the chitosan chains,
without any significant change in DD, led to membranes
with lower water vapour permeability and swelling in water.
This significant effect is probably related to the increasing
excluded volume effects with increasing molecular mass,
contributing to the formation of a more effective packing of
polymer chains within the membrane matrix and a lower
degree of interstitial space in the case of those membrane
networks formed from lower Mw chitosan chains. However,
this decrease impairs mechanical properties as a result of
decreasing entanglement density and crosslinking degree and
formation of a looser network [183].
Table 8. Influence of Chitosan DD and Mw on Fat Binding and Fat Metabolism

Physico-Chemical Property Effect In Vitro Effect In Vivo
DD electrostatic force between chitosan and
fatty and bile acid
plasma cholesterol
LDL
HDL
Mw adsorption to lipid droplets
adsorption to droplet surface of lipase
body weight gain
adsorption and blood distribution
liver total lipid and cholesterol
LDL: low density lipoprotein.
HDL: high density lipoprotein.
The use of chitosan coating as a protective barrier to ex-
tend the storability of many fruits and vegetables has been
widely documented [184-186]. The latest reports consider
the low Mw chitosan. The effect of coating Murcott tangor
with low and high Mw chitosan has been investigated. This
study showed that low Mw chitosan retarded ripening, water
loss and decay. The coated samples exhibited greater anti-
fungal resistance than thiabendazole [178]. Similar results
were obtained with the low Mw with sliced red pitayas
[187].
J eon et al. compared the preservative efficacy of different
viscosity chitosans in coated herring and Atlantic cod. This
study demonstrated the potential of chitosan as a preserva-
tive coating in reducing or preventing moisture loss, lipid
oxidation, and microbial growth, the higher viscosity (360
cP) chitosan exerting a better preservative effect in both fish
model system s [188]. The effect of the DD and the Mw has
been reported in the preservation of fish fillets. Presoaking
the samples with a high DD chitosan solution extended the
shelf life from 5 to 9 days. Chitosan showed stronger activity
against bacteria rather than against fungi [56].
The coating of eggs for shelf life improvement with chi-
tosan has attracted much interest in recent years. Chitosan
may offer a protective barrier against the transfer of carbon
dioxide and moisture through the eggshell. This barrier was
also effective in keeping a high Haugh unit and yolk index,
whose decrease is caused mainly by diffusion of water from
the albumen. Only one of the groups involved in these stud-
ies has reported the comparison of different Mw chitosans.
The results showed that lower Mw chitosan presented the
strongest bactericidal effect and best kept the internal quality
of eggs [189].
7.3. Emulsifying Agent
Chitosan produces w/o/w emulsions without adding any
surfactant, because this biopolymer is composed of a mixture
of molecules with different DD: some less deacetylated
molecules may stabilize the water droplets inside the oil
drops, while the hydrophilic ones stabilize the oil drops in
the multiple emulsion formed [190].
The influence of the DD of chitosan on the emulsification
of sunflower oil has been studied in HCl solutions. The re-
sulting distribution was unimodal at low DD (75%) and high
DD (95%) for all used concentrations. At intermediate DD,
distribution was unimodal only when the most concentrated
solution was used. When chitosan concentration increased,
emulsion viscosity as a function of time was more stable
[191].
Laplante et al. studied the emulsion stabilizing properties
of various chitosans in the presence of whey protein isolate
(WPI) [192]. A low deacetylated chitosan seemed better for
stabilisation of the emulsion. They reported that there is a
positive correlation between the increase of stability against
syneresis and the increase of viscosity of chitosan (Table 9).
The most unfavourable effect from a low Mw preparation on
stability is mainly explained by a loss of interfacial coad-
sorption efficiency. When studying other variables such as
pH, ionic strength and the WPI/chitosan ratio, a low deacety-
lated chitosan displayed better behaviour, showing the pre-
dominance of electrostatic interactions in the interfacial sta-
bilisation.
Table 9. Influence of Chitosan DD and Viscosity on Emulsify-
ing Activity

Physico-Chemical Property Effect on Emulsifying Activity
DD stabilization of emulsion
Viscosity stability against syneresis
Emulsions stabilized by surfactant-chitosan membranes
have been shown to have better stability to pH, ionic
strength, thermal processing and freezing than emulsions
stabilized by surfactant alone, which was attributed to the
increase in electrostatic and steric repulsion between the
droplets [96,193]. The influence of the molecular character-
istics of chitosan on the properties of oil-in-water emulsions
has been studied. The - potential and mean diameter of the
particles in the secondary emulsions was not strongly influ-
enced by chitosan Mw; however, with the lowest DD (40%)
the fraction of droplets that were aggregated was considera-
bly lower [194].
Table 9 summarized the effect of chitosan Mw and DD
on emulsions.
8. APPLICATION OF CHITIN AND CHITOSAN IN
BIOCATALYSIS
Immobilization was firstly defined as the process in
which the enzyme is confined in a definite position thus ren-
dering an insoluble form which retains the catalytic activity
and can be reused several times. Later, this definition was
extended to be defined as the process which includes not
only enzymes but also cells, organelles and so on. There are
four principal methods available for immobilizing enzymes
and cells: adsorption, covalent binding, entrap-
ment/membrane confinement and cross-linking [195].
Chitin and chitosan have been widely used as supports
for enzyme and cell immobilization due to their appropriate
characteristics. The extraction of chitin from shells and the
subsequent deacetylation of chitin to produce chitosan is a
relatively low cost process. Moreover, the raw material is a
by-product of the seafood processing industry, the produc-
tion of chitin and chitosan being a means to reduce these
waste products. Both supports present appropriate density,
mechanical stability and rigidity.
Chitin has been used mainly for the immobilization of
enzymes and to a lesser extent to immobilize cells. On the
other hand due to its higher versatility, chitosan has been
used to immobilize not only enzymes but also cells, mainly
by entrapment and membrane confinement. Table 10 sum-
marized the use of chitin and chitosan for cell immobiliza-
tion [196-206].
Several studies covering the field of the immobilization
of enzymes on chitin and chitosan have been published [207-
209] but the effect of chitin and chitosan characteristics is
not issued. In general, not many specific studies related to
the effect of the properties of chitin and chitosan on this field
are found in the literature. The vast majority of the research
focuses on the method of immobilization and its effect on the
properties of the biocatalyst without taking into account that
chitin and chitosan samples are available with a wide range
of Mw and DD. In this part of the review we intend to show
the relationship between chitin and chitosan properties and
the behaviour of the immobilized biocatalyst.
8.1. Immobilization of Enzymes and Cells on Chitin and
Chitosan (Adsorption and Covalent Binding)
In immobilization by adsorption, the enzymes or cells are
bound to the carrier material via reversible surface interac-
tions. The forces involved are van der Waals forces, ionic
and H-bonding interaction as well as hydrophobic forces.
Due to the low amount of free amino groups on its surface,
chitin can be considered a neutral support and there is a low
possibility of interaction through H-bonding or ionic forces.
As the amount of free amino groups increase the ionic and
H-bonding forces achieve more relevance. When attaching to
non-charged supports such as chitin, proteins showed their
maximal adsorption at their isoelectric point [210]. On the
other hand, when the support is charged the maximal interac-
tion occurs when both the enzyme and the support exhibit
opposite charge. Chitosan is a positively charged polymer at
pH lower than its pKa (6.4), in these conditions negatively
charged proteins bind easily to chitosan.
The adsorption of cells on chitin and chitosan has also
been studied. Strand et al studied the effect of the chemical
composition and Mw of chitosan on the adsorption and floc-
culation of E. coli cells. These authors found that adsorption
increases with the pH but decreases with the Mw of chitosan.
Chitosan samples with a high DD showed the best adsorp-
tion behaviour [197].
The covalent immobilization involves the formation of a
covalent bond between the enzyme/cell and the support ma-
terial. The bond is formed between functional groups on the
carrier and the enzyme (amino, carboxyl, hydroxyl and sul-
phydryl groups). Through chemical modification, functional
groups on the carriers can be altered to create new interac-
tions between the enzyme and the support. In chitin and chi-
tosan the most usual process for the immobilization of the
enzymes and cells is the immobilization through a bifunc-
tional dialdehyde (most frequently glutaraldehyde). The den-
sity of free amino groups on the surface of the support will
determine the amount of interaction points between the en-
zyme and the support. When using chitin as support, the
number of free amino groups on the support will be low and
then each enzyme molecule may be attached to the support
in a single point. On the other hand, when chitosan is used as
support each enzyme may be linked to the support through
several residues. In these conditions a multipoint immobili-
zation is achieved (Fig. (5)).
In our laboratory, a crude cell extract from Agrobacte-
rium radiobacter, containing D-hydantoinase and D-
carboamylase activities to produce D-amino acids, was im-
mobilized on different chitin and chitosan samples by ad-
sorption, covalent binding and membrane confinement [211-
213]. When the extract was immobilized by adsorption, the
results showed that the activity of the immobilized deriva-
tives on chitin were around 2-fold higher than the ones im-
mobilized on chitosan (Table 11). On the other hand, when
the extract was immobilized by covalent binding on chitosan
no activity was detected. In this particular case, the extract
immobilized on chitin showed half of the activity immobi-
lized by adsorption.
These results showed the effect of the DD on the biocata-
lyst activity. When the biocatalyst was immobilized by ad-
sorption on chitosan, the low activity was related to the pI of
the catalytic enzymes of the extract. The pIs of D-
Table 10. Examples of the Use of Chitin and Chitosan as Support for Immobilization of Cells

Cells Method Support Ref
Rhodococcus sp Adsorption Chitin [196]
E. coli Adsorption Chitosan [197]
Several microorganism Entrapment -Chitin gels [198]
Agrobacterium radiobacter Covalent binding Chitin and chitosan powders [199]
Pseudomonas sp Adsorption Chitosan agar-alginate beads [200]
Pseudomonas putida Membrane confinement Chitosan beads croslinked with TPP [201]
Several microorganism Membrane confinement Chitosan beads [202]
Saccharomices cerevisiae Membrane confinement Chitosan- carboxymethil celulose [203]
Saccharomyces cerevisiae Covalent binding Globular chitosans activated with glutaraldehyde [204]
Hepatocyte spheroids and hepatocytes Membrane confinement Alginate-chitosan polyelectrolyte complex [205]
Sporidiobolus salmonicolor Membrane confinement Chitosan capsules crosslinked with gloxal and diso-
dium sulphate
[206]

Fig. (5). Covalent immobilization of enzymes on chitin/chitosan. a)
Low free amino group density (Single point immobilization) and b)
high free amino group density (multipoint immobilization).
hydantoinase and D-carboamylase from Agrobacterium have
been reported to be 6.5 and 5.5 [214,215]. These values are
close to the pKa of chitosan and thus the interaction between
the enzymes and the support are not favoured. In the case of
covalent immobilization, the absence of the activity can be
related to conformational changes due to the multipoint im-
mobilization as well as irreversible interactions between
catalytic residues of the enzyme and the support [213].
A new green bean (Phaseolus vulgaris) tissue homogen-
ate-based biosensor was developed for the square-wave volt-
ammetric determination of caffeic acid in white wine. The
biosensor was constructed by immobilization of green bean
tissue homogenate, as a source of peroxidase, in a chemi-
cally crosslinked chitin matrix with epichlorohydrin and glu-
taraldehyde that was incorporated in a carbon paste elec-
trode. Two samples of chitin, a commercial one from Sigma
and a homemade one from squid pens were tested. The squid
pen samples were chosen as the most suitable due to a higher
DD (91.6 vs 86.8) [216].
Another property of the samples that shows a great effect
on the activity is the ash content of the supports. When the
extract was immobilized on lobster chitin and chitosan no
activity was detected. Synowiecki et al. studied the immobi-
lization of -amylase on several chitin samples by adsorption
and covalent binding; in both cases, the enzyme immobilized
on supports with high ash content showed lower activity
[217]. On the other hand, we have used lobster chitin sam-
ples with high ash content to immobilize tyrosinase and -
chymotripsin and in both cases, the biocatalyst showed good
catalytic properties [218,219]. In this context, it is clear that
a deeper study of the effect of the ash content should be con-
ducted to clarify these results.
Chitin crystallinity is another aspect to keep in mind
when choosing the best chitin for a catalytic purpose. Since
-chitin is a more hydrated structure than -chitin, chitin
samples with high -structure content are more hydrophilic
supports. Moreover, due to much weaker intermolecular hy-
drogen bonding ascribable to the parallel arrangement of the
main chains, -chitins are more reactive than -chitins [220].
Enzymes are able to catalyze reactions not only in aqueous
media but also in organic media. In these processes the water
content in the microenvironment of the enzymes is basic to
the catalytic process.
When -chymotrypsin was immobilized by covalent
binding on several chitin samples the derivative immobilized
on squid chitin (-chitin) showed the highest hydrolytic ac-
tivity in organic media due to the higher water content in the
microenvironment of the enzyme [219]. Due to the higher
porosity of krill chitin (-chitin) when compared to other
sources, this chitin was selected as the most appropriate sup-
port for modification with carbon disulfide [221]. This de-
rivative has both positively charged amino groups and nega-
tively charged dithiocarbamino groups showing better cata-
lytic properties than control chitin when several enzymes
were immobilized by adsorption. Squid pen chitin (-chitin)
and crab chitin have been used to prepare thin films to im-
mobilize several oxidases to prepare a biosensor. The struc-
ture of the -chitin sample was more favourable than the -
chitin sample [222].
Laccase has been conjugated with chitosan of several
Mw with a carbodiimide reaction. The effect of the Mw of
chitosan on the activity loss during conjugation was studied.
Small chitosan molecules gave high residual activity. The
conjugated laccase exhibited a high stability in the following
repeated phase changes and had the same temperature and
pH profile as those of free laccase. Compared to free laccase,
the conjugated laccase had a similar affinity (K
m
), but re-
duced turnover (k
cat
) that was adversely affected by the in-
crease of the molecular mass of chitosan [223].
The source of the support also affects the properties of
the biocatalyst. A lipase has been immobilized on chitosan
films prepared from mycelia of S. racemosum and from a
crustacean source using glutaraldehyde as a bifunctional
agent. The lipase immobilized on the crustacean chitosan
showed higher activity that the one immobilized on the my-
celia sample. However, after four cycles the remaining activ-
ity of the lipase immobilized on mycelia chitosan was higher
[224].
8.2. Entrapment of Biocatalysts on Chitosan and Chito-
san Derived Systems
In encapsulation and entrapment the enzyme or cells are
free in solution, but restricted in movement by the lattice
Table 11. Chitin and Chitosan as Support for the Immobilization of a Crude Cell Extract by Adsorption and Covalent Binding

Sample Source DA Ash, % Crystallinity p-HPG Yield, %
1
p-HPG Yield, %
2

Chitin Shrimp (Ploeticus mlleri) 0.96 0.33 18 8.1
Chitin Shrimp (Penaeus caramote) 0.95 0.62 14 8.0
Chitin Lobster (Palinurus vulgaris) 0.96 12.25 0 0
Chitin Squid (Dosidicus Gigas) 0.91 0.48 16 ----
Chitosan Shrimp(Penaeus caramote) 0.33 0.22 ---- 9 0
Chitosan Lobster(Palinurus vulgaris) 0.61 8.25 ---- 0 0
Chitosan Crab (Paralomis granulosa) 0.16 0.89 ---- 9 0
Chitosan Crab Commercial 0.09 0.15 ---- 10 0
1
Immobilization by adsorption,
2
Immobilization by covalent binding.
DA: acetylation degree.
p-HPG: p-hydroxyphenylglycine.
structure of a gel. The porosity of the gel lattice is controlled
to ensure that the structure is compact enough to prevent
leakage of the enzymes or cells, while allowing free move-
ment of substrate, cofactors, products and nutrients when
necessary.
Alsarra et al. have studied the effect of the Mw and DD
of chitosan on the activity of lipase encapsulated in chitosan-
TPP capsules [225]. These authors found that chitosan with
high Mw and DD improve lipase loading and lessen the re-
lease of the entrapped lipase. Moreover, they developed
equations which can predict the effect of the Mw and DD on
entrapment efficiency and other parameters of interest.
Alginate-chitosan polyelectrolyte complexes can be pre-
pared by different methodologies. Among them, the one-step
procedure and two-step procedures have been used to encap-
sulate proteins, mainly with the purpose of preparing drug
delivery systems. However, this method can also be used to
encapsulate enzymes with a catalytic purpose [226, 227]. In
our laboratory, a crude cell extract from Agrobacterium rb
has been encapsulated by using both methods. The effect of
the Mw and DD of chitosan on the properties of the biocata-
lyst was studied. The amount of encapsulated protein was
around 60% and did not depend on the chitosan characteris-
tics. In all cases, a negligible release was observed so the
biocatalyst can be used in both long term process and in
batch. The effect of the chitosan characteristics on the me-
chanical stability of the capsules is shown in Table 12. It is
clear that the stability of the capsules is greatly dependent on
the Mw of chitosan being unaffected by the DD (Fig. (6)).
The ability of the biocatalyst to produce p-
hydroxyphenylglycine was also tested. As can be seen in
Table 13, the DD of chitosan negatively affects the produc-
tion of the amino acid while the Mw showed a lighter effect
on it.
Alginate capsules coated with chitosan have also been
used to protect urease from -chymotrypsin and other prote-
ases. The effect of the Mw of chitosan on the retention activ-
ity of urease after treatment with -chymotrypsin was stud-
ied. The best results were observed with the medium Mw
chitosan with a retention activity of 48% while the low and
high Mw showed a lower retention of 20 and 25%, respec-
tively. Activity retention of urease encapsulated in alginate
coated capsules with medium Mw chitosan after treatment
with trypsin and proteinase K was 44 and 73% respectively
[228].
Table 13. Effect of Chitosan Characteristics on the Production
of p-HPG

CHITOSAN
Mw,
DD
p-HPG
Yield,(%)
1

p-HPG
Yield,(%)
2

816 0.90 68 73
495 0.90 68 63
75 0.90 63 60
563 0.70 38 30
1
One step procedure.
2
Two steps procedure.
p-HPG: p- hydroxiphenylglycine.
Alginate mixed chitosan capsules have been used to im-
mobilize carbonic anhydrase (Mw 30 kDa) for a biocatalytic
purpose. The effect of the Mw of chitosan on the enzyme
retention was studied. The best enzyme retention was
achieved with the medium Mw chitosan [229]. Tannase has
been encapsulated in chitosan crosslinked TTP capsules as
well as in alginate coated chitosan capsules for the hydroly-
Table 12. Mechanical Stability of the Alginate-Chitosan PEC
Capsules Loaded with Extract. Burst Assay (N=100)

CHITOSAN
Mw, kDa DD
Stability, (%)
1
Stability, (%)
2

816 0.90 30 100
495 0.90 100 100
75 0.90 0 100
563 0.70 100 100
1
One step procedure.
2
two step procedure.
PEC: polyelectrolyte complex.

Fig. (6). Alginate-chitosan polyelectrolyte complexes. a) High molecular weight chitosan piles on the capsule surface b) Low Molecular
weight chitosan gets into the alginate network.
sis of tea tannins [230]. A high Mw and a low Mw chitosan
were used. The release of the enzyme from the alginate-
chitosan capsules was studied. After three days, the enzyme
encapsulated using the high Mw chitosan showed a higher
release than the small one (83% vs 28%).
9. WASTE WATER TREATMENT
The chemical contamination of water from a wide range
of toxic products such as metals, aromatic molecules, dyes
and so on, is a serious environmental problem owing to the
latters potential human toxicity. The use of low-cost poly-
mers such as chitin and chitosan to remove water pollutants
is of great interest [231].
Chitin and chitosan have been used to remove a great
variety of water pollutants [232]. Guibal and co-workers
have extensively studied the effect of chitosan properties on
the adsorption of metals, dyes and organic compounds
among others [233-235]. The use of chitosan based material
to remove anionic dyes has been recently reviewed by Crini
and Badot [236]. This work includes a specific mention of
the effect of chitosan characteristics on the adsorption proc-
ess.
The coagulation-flocculation process as well as the ad-
sorption process depends on the DD, chitosan being more
efficient than chitin to remove metal ions [233, 237], PCBs
[238] and anionic dyes [232]. On the other hand, chitin was
more efficient than chitosan in removing polycyclic aromatic
hydrocarbons from petrochemical wastewater [239]. When
comparing chitosan samples with different DD for the ad-
sorption of an azo dye the sample with the higher DD
showed the highest efficiency [240]. However, in some cases
this rule does not work.
Guibal and coworkers have suggested that rather than the
deacetylation degree the parameter that controls these proc-
esses is the accessibility of the amino groups to water and
pollutants [233-235]. This accessibility is mainly controlled
by the crystallinity of the samples, which depends on the
origin of the sample as well as the treatment during extrac-
tion.
Zhang and Schiewer have also studied the effect of the
deacetylation degree and crystallinity of chitin and chitosan
samples (DD from 10 to 90%) on the adsorption of As (V)
[241]. Their results also support the role of crystallinity in
the adsorption of As (V). Wong et al. studied the adsorption
of five acidic dyes on chitin and chitosan samples [242].
They suggested that the adsorption mechanism involves a
one-step process of dyes complexing with the free amino
groups. When varying the DD from 52% to 97% a decrease
in the dye adsorption capacity was observed suggesting that
more amorphization may cause changes in the internal struc-
ture of chitosan and reduce the adsorption capacity.
Moreover, Kurita et al. found that metal adsorption rate
for samples with the same DD was higher when homogene-
ous hydrolysis was used to prepare the chitosan samples,
which indicates that the distribution of the acetyl groups
along the chains also affects the adsorption process [243].
Huang et al. have found that the coagulant efficiency of
chitosan also depends on the Mw of the sample. Samples
with the same Mw increase their coagulant efficiency by
increasing DD but when the Mw of the sample was increased
a reduction of the coagulant efficiency was reported even
when DD was higher (86 vs 77%) [244]. Bought et al. com-
pared several chitosan samples with different Mw and vis-
cosity and found that higher values of Mw were predictive of
greater effectiveness for coagulation but the effectiveness did
not correlate linearly with Mw [245].
Chitosan has been used as adsorbent, coagulant and bac-
tericide in the treatment of aquaculture wastewater. The
higher the DD, the higher the removal of organic com-
pounds, inorganic nutrients (nitrates and phosphates) and
pathogen was. On the other hand, the higher Mw chitosan
showed a higher reduction of turbidity, suspended solids,
BOD and COD while the lower Mw was very effective in
the reduction of inorganic nutrients [174,246].
The effectiveness of chitosan in coagulating and floccu-
lating organic suspensions at pH close to neutrality and low
ionic strength (distilled water) is improved by using high DD
and low Mw chitosan samples [234]. On the other hand, the
effect of DD and Mw decreased at acidic pH and higher
ionic strength (tap water), the effectiveness of the process
being almost unaffected by chitosan conditions. When con-
centrated suspensions of bentonite were adsorbed on chito-
san in either tap water or demineralized water at either pH 5
or 7, efficiency was higher when the Mw of the chitosan
samples was higher, though efficiency tended to level off
when the Mw of chitosan exceeded 10kDa [235].
Alginate-chitosan polyelectrolyte complexes using dif-
ferent Mw and DD chitosan samples were used to recover
soluble proteins from surimi wash water. Protein recovery
from aqueous processing streams by chitosanalginate com-
plexes has been proposed to occur by mechanical entrapment
and electrostatic interaction with the complex. The effect of
DD and Mw of chitosan was tested. However, no correlation
was found [247].
Apart from DD and Mw, the purity (protein and pigment
content) of the samples is another aspect that affects the ef-
fectiveness of chitinous materials in the field of waste water
treatment since it affects the accessibility of the free amino
groups to the pollutants. From these results, it can be con-
cluded that the choice of a chitinous sample for a specific
pollutant will depend on several factors, such as the type of
pollutant, polymer characteristics (mainly DD, crystallinity
and Mw) and the characteristics of the water (pH, ionic
strength). However, as a general rule chitosan is preferred
over chitin in the vast majority of applications. Moreover,
chitosan samples with low crystallinity and high DD are
generally preferred.
10. NEW APPLICATIONS OF CHITIN AND CHITO-
SAN
Apart from the aforementioned applications of chitin and
chitosan which have been developed in recent decades, new
applications that have not been previously reviewed have
appeared in the last few years. Although not much informa-
tion can be found with regard to these applications we have
tried to update the knowledge on the relationship between
physicochemical properties and function.
10.1. Imprinted Chitosan-Based Matrixes
Molecularly imprinted polymer (MIP) represents a new
class of materials that have artificially created receptor struc-
tures. This potential technology is a method for making se-
lective binding sites in synthetic polymers by using a mo-
lecular template. MIPs have steric and chemical memory
toward the template and hence could be used to rebind it
(Fig. (7)).
As can be seen in Table 14, chitosan imprinted matrixes
have been prepared with different purposes by using differ-
ent chitosan samples [248-265].
In general, samples with DD 90% and wide range of
Mw have been used to prepare chitosan imprinted materials.
As far as we know, there is only one paper studying the ef-
fect of chitosan Mw (92, 47 and 29 kDa) on the rebinding
properties of molecular imprinted chitosan matrixes [255]. In
this case, the sample with 47 kDa achieved the best rebind-
ing capacities.
It is well known that the number of recognition sites in
MIPs increases with network density. The crosslinking den-

Fig. (7). Schematic representation of imprinted chitosan-based matrix preparation.
Table 14. Preparation of MIP Chitosan with Different Purposes

Matrix CS Characteristics Crosslinker Template Application REF
Cs hydrogels DD 94%,30 kDa Glu DBT Fuel desulfurization [248]
Cs hydrogel DD 90%, 78 kDa Genipin O-xylene Waste water treatment [249]
CS Non data ECH, GLU PFOS Waste water treatment [250]
Cs DD 90%, 521 ml/g ECH Hemoglobin Protein binding [251, 252]
Cs, binary
xilosanes
DD 98% 6*10
4
g/mol ECH, form BSA Protein binding [253]
CS , nylon mem-
branes
DD 98%, 1*10
5
g/mol GPTMS PEG Protein binding [254]
CS, acrylamide DD 90%, 92,47,29 kDa MBA BSA Protein binding [255]
CS, acrylamide DD 90%, 504 kDa MBA Hb Protein binding [256]
Cs TiO2 DD 90% ECH Ni, Cu Metal Recovery and photodegradation [257]
Cs fungi myce-
lium
DD 90% ECH Ni Metal recovery [258]
Cs, fungi myce-
lium, TiO2
DD 90% ECH Ni Metal Recovery and photodegradation [259]
CS resin DD 90% ECH Ni Metal recovery [260]
Cs silica gel DD 98%, 8*10
5
g/mol GPTMS PEG, sucrose Metal recovery [261]
Cs , fungi myce-
lium, TiO2
CS 90% ECH Ni Organic compound degradation and
metal recovery
[262]
Cs silica gel DD 98%, 6*10
4
GPTMS Cd Cd recovery [263]
CS, acid metac-
rilic
DD 90%, 521ml/mg ECH quercetin Flavonoids recovery [264]
Cs DD 92% GPTMS L-Phe Quiral resolution [265]
ECH: epichlorohydrin GLU: Glutaraldehyde GPTSM -glycidoxypropyltrimethoxysilane; MBA: metilenbisacrilamida; Form: Formaldehyde DBT: Dibenzothiophene sulfone.
PFOS: Perfluorooctane sulfonate. BSA: bovine serumalbumin. L-Phe : L-phenylalanin. PEG: Polyethylene glycol.
sity is influenced by various parameters being favoured by
high DD and Mw. Since crystallinity and surface area con-
trol the accessibility of the free amino groups to the chemical
reagents, these parameters are expected to have an impact on
the behaviour of the matrixes.
10.2. Chitosan-Metal Nanocomposites
As mentioned above, chitosan has high affinity for metal
ions. This property has been used to prepare metal-chitosan
nanocomposites with potential applications in several fields,
such as biomedicine, catalyst, electronics, non-linear optics
and so on.
Traditionally, metal ions were reduced by chemical
agents, UV-light in the presence of chitosan which stabilized
and controlled the size of the nanoparticles due to the pres-
ence of free amino groups [266]. Taking into account that
some sugars are able to reduce metal ions, chitosan has been
used to reduce the metal ions in situ without using other
reductants. Gold nanoparticles have been prepared in acidic
media in the presence of chitosan at 55C [267]. Twu et al.
prepared silver nanoparticles using chitosan (1240 kDa,
DD=87%) suspensions in basic medium at high temperature
and suggested that low Mw chitosan degradation products
may supply electrons and function as a reducing agent [268].
Murugadoss and Chattopadhyay have also prepared a silver-
chitosan nanocomposite in basic medium at high temperature
using a high Mw chitosan from Sigma (DD=75%, no data on
Mw) [269]. A more exhaustive characterization of the sys-
tem was carried out and these authors propose that free
amino groups as well as hydroxyl groups are involved in
Ag+reduction. Moreover, Ag nanoparticles are adsorbed on
the surface of the polymer due to the free amino groups and
prevent further aggregation controlling the size of the
nanoparticle.
Although the aforementioned authors indicated in their
studies that the silver reduction only occurred in a basic me-
dium, Wei and Qian have prepared silver and gold chitosan
nanocomposites in acidic medium by using a chitosan sam-
ple with DD higher than 85% and viscosity higher than
200.000 cps. [270]. These authors suggested that free amino
groups are bound to Ag nanoparticles during the reduction
and the reduction of metal is due to oxidation of hydroxyl
groups. Due to a not proper characterization of the samples a
direct comparison is not possible; but it seems that the reduc-
tion of silver ions in basic media depends on the Mw of chi-
tosan samples. Silver nanoparticles have also been prepared
by -radiation of a chitosan solution in acidic media. The
authors have suggested that the stabilization of Ag nanopar-
ticles is due to the presence of depolymerized chitosan due to
radiation [271].
Recently, Wu et al. have prepared gold nanoplates by
reducing gold with a chitosan sample with a Mw of 50 kDa
[272]. These authors found that by increasing the chitosan
(Mw 1000 kDa), the final gold products were mainly com-
posed of spherical gold nanoparticles and a small portion of
sub-micro-sized gold nanoplates which indicates that chito-
san Mw controls the morphology of the system. Moreover,
Yang et al. have prepared gold 2D nanoparticles chains by
using a low Mw chitosan (5000 Da) [273]. All these results
point to a key role of chitosan characteristics, namely DD
and Mw, in the synthesis of metal nanoparticles as well as in
their morphology (Fig. (8)).
11. CONCLUSION
As we have shown in this review, chitin and chitosan
present a great variety of properties, allowing them to have a
large number of applications, but, at the same time, the very
complex behaviour of these polymers is difficult to control.
These polymers have an intrinsic variability due to their
natural origin. Moreover, depending on the manufacturing
process, the properties of samples from the same source
change. In general, a poor characterization of the polymers is
carried out which makes it very difficult to compare results
and to establish relationships between the physiological be-
haviour of chitin and chitosan and their properties. However,
from data in the literature it is possible to give general rec-
ommendations regarding the properties of chitin and chito-
san for a specific application, this being the main contribu-
tion of this review to the field of study of chitin and chitosan.
These recommendations are shown in Table 15.
ACKNOWLEDGEMENTS
This study was conducted within the framework of the
MAT 2000-0037-P4-03, MAT2004-03982 and MAT2007-
63753 projects from the Spanish Ministry of Education.

Fig. (8). Effect of chitosan molecular weight on the morphology of gold nanoparticles. (A) nanoplates (1000kDa), (B) single nanoparticles
(50kDa) and (C) 2D chains (5 kDa).
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Table 15. General Recommendations for the Use of Chitin and Chitosan in Several Applications

Application General Recommendations
Wound healing
High DD chitosan preferred over chitin
Low Mw samples (oligomers)
Drug delivery systems
High DD
High Mw
Gene Delivery
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Received: March 09, 2008 Revised: J uly 21, 2008 Accepted: November 20, 2008

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